Regiochemical Analysis of the ProTide Activation Mechanism

ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes in vivo enzymatic and spontaneous chemical transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P–N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P–O versus C–O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in 18O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and 31P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.


■ INTRODUCTION
−3 Originally developed to improve treatment for HIV, ProTides have become a leading therapeutic method for other viral infections. 4,5ProTides consist of a phosphoramidate (P−N) core coupled with a nucleoside analogue, a phenol, and an L-alanyl carboxylate ester. 6−9 The structures of these compounds are highlighted in Figure 1.
−14 The next step is hypothesized to be the spontaneous attack of the phosphorus core by the newly formed carboxylate, resulting in the release of phenol and formation of a 5-membered mixed anhydride cyclic intermediate. 6,12Recent mass spectrometry experiments have provided support for the 5-membered cyclic intermediate. 15,16he mixed anhydride cyclic intermediate has been proposed to be spontaneously attacked by water to form a linear phosphoramidate product. 6,17,18The linear phosphoramidate product from the putative cyclic intermediate could result from the attack of water at the phosphate or carboxylate ester.In either case, the product is the same.−21 The site of hydrolysis of the proposed cyclic intermediate has not been determined (P−O vs C−O bond cleavage). 6,15,16ere, we have further interrogated the mechanism of ProTide activation using ESI-mass spectrometry and 31 P NMR spectroscopy when the products are formed in oxygen-18 labeled water.The results from the oxygen-18 labeling conclusively demonstrate that the cyclic intermediate is hydrolyzed via the attack of water at the phosphorus center rather than the carboxylate.

■ MATERIALS AND METHODS
Materials.General lab supplies were purchased from SigmaAldrich and VWR. S. cerevisiae carboxypeptidase Y was purchased from SigmaAldrich and prepared according to the manufacturer's specifications. 18O-labeled water (≥97%) was purchased from Medical Isotopes Inc.

Synthesis of ProTide Analogue 1a.
A stirred solution of ethyl dichlorophosphate (96%, 0.78 mL, 6.82 mmol, 1.1 equiv) in 20 mL of anhydrous dichloromethane was cooled to −78 °C.Phenol (89%, 0.65 mL, 6.0 mmol, 1 equiv) was added, followed by the dropwise addition of triethylamine (6.0 mmol, 0.84 mL, 1.0 equiv).The reaction mixture was allowed to warm to room temperature and then stirred for 4 h.The reaction mixture was cooled to −78 °C, L-alanine isopropyl ester hydrochloride (1.04 g, 6.2 mmol, 1.0 equiv) was added, and then triethylamine (1.7 mL, 12.0 mmol, 2.0 equiv) was added dropwise.The reaction mixture was allowed to warm to room temperature (23 °C) and stirred for an additional 18 h.The mixture was filtered and concentrated, and the residue was purified by silica gel column chromatography (4:3 and 1:1, hexanes/ethyl acetate mixtures).The final product (compound 1a) was a colorless oil as a mixture of two diastereomers yielding 0.68 g (35%).
Spectroscopic analysis: Synthesis of ProTide Analogue 1b.L-Alanine isopropyl ester hydrochloride (1.04 g, 6.2 mmol, 1.0 equiv) was suspended in 20 mL of anhydrous dichloromethane and cooled to −78 °C.Ethyl dichlorophosphate (96%, 0.78 mL, 6.82 mmol, 1.1 equiv) was added, followed by the dropwise addition of triethylamine (1.7 mL, 12.4 mmol, 2.0 equiv).The cooling bath was removed, and stirring continued for an additional 3 h at room temperature.The reaction mixture was then cooled to 0 °C, and p-nitrophenol (0.77 g, 5.6 mmol, 0.9 equiv) was added, followed by the addition of triethylamine (0.85 mL).The reaction mixture was allowed to stir at room temperature (23 °C) for 18 h.The mixture was filtered, concentrated, and the residue purified by silica gel column chromatography (4:1, 2:1, and 1:1 hexanes/ethyl acetate mixtures).The final product was a colorless oil as a mixture of diastereomers yielding 0.65 g (29%).The structures of compounds 1a and 1b are presented in Figure 3.   UV−vis Spectra.Spectral analyses of compounds 1a and 1b were performed using a SpectraMax 384 UV−vis spectrophotometer from Molecular Devices.Compounds 1a and 1b were resuspended and diluted with dimethylformamide (DMF).The UV−vis spectra of each compound were obtained before and after the addition of carboxypeptidase Y to initiate the hydrolysis of the isopropyl carboxylate ester.Reactions (1.0 mL) contained 50 mM triethanolamine-HCl (pH 8.0) and 1.0 mM of compound 1a or 100 μM of compound 1b (1% DMF).Reactions were initiated by the addition of 1.0 μM carboxypeptidase Y at 25 °C.
The ability of carboxypeptidase Y to differentially hydrolyze the two diastereomers of compounds 1a and 1b was tested using 31 P NMR spectroscopy.Enzyme assays (500 μL) were conducted in 100 mM triethanolamine-HCl (pH 8.0) using either 4.0 mM 1a or 1b (2% DMF) at 30 °C.Reactions were initiated by the addition of carboxypeptidase Y and 31 P NMR spectra were taken periodically.
Positional Hydrolysis of Putative Cyclic Intermediate.Reactions (100 μL) were conducted in 90% 18 O-labeled water, 50 mM triethanolamine-HCl (pH 8.0), and either 4.0 mM 1a or 1b (2% DMF).Reactions were initiated by the addition of 1.9 μM carboxypeptidase Y and incubated at 25 °C for 1 h.Samples were analyzed by electrospray ionization mass spectrometry (ESI-MS) to determine the extent of incorporation of 18 O to the ultimate reaction product.Control reactions were conducted in 16 O-water.
Additional reactions (500 μL) were also conducted in 90% 18 O-labeled water, 50 mM triethanolamine-HCl (pH 8.0), and 4.0 mM of either compound 1a or 1b (2% DMF).Reactions were initiated by the addition of 1.9 μM carboxypeptidase Y and incubated at 25 °C for 1 h.After the reactions were complete, 55 μL of D 2 O was added to the solution and the sample analyzed by 31 P NMR spectroscopy.Control reactions were carried out in 16 O-water for both compounds.
Rate of Cyclization.Reactions (500 μL) were performed in 100 mM triethanolamine-HCl (pH 8.0), 4.0 mM 1a (2% DMF), and 10% D 2 O at 30 °C.The reaction was initiated by the addition of 3.9 μM carboxypeptidase Y and monitored using 31 P NMR spectroscopy at 6 min intervals.The rate of product formation and substrate decay were calculated using eqs 1 and 2, respectively.Additionally, the rate of cyclization at 25 °C was determined by UV−vis spectroscopy using 0.40, 0.60, 0.80, and 1.0 μM carboxypeptidase Y in 50 mM triethanolamine-HCl (pH 8.0) starting from 400 μM of compound 1a.These reactions were monitored at 271 nm, and the data were fit to eq 1.
Data Analysis.Time courses were processed using SigmaPlot 11.0.Data for the hydrolysis reactions and the determination of the kinetic constants were analyzed as follows.The time courses for the carboxypeptidase Y catalyzed hydrolysis reactions were fit to a single exponential (eq 1) where k is the first-order rate constant for the hydrolysis of substrate.For reactions in which the stereoselectivity for enzymatic hydrolysis was biphasic, the entire time course was fit to a double exponential (eq 3), where k 1 is the rate constant for hydrolysis of the first diastereomer and k 2 is the rate constant for hydrolysis of the slower diastereomer.
■ RESULTS AND DISCUSSION Synthesis of ProTide Analogues.Two pairs of diastereomeric ProTide analogs were synthesized as probes of the chemical reaction mechanism for prodrug activation.Both pairs of diastereomers contain an ethoxy substituent to mimic the modified nucleoside, and an L-alanyl isopropyl ester connected to the phosphorus core via an amide linkage.Compound 1a contains a phenolic leaving group, whereas compound 1b is substituted with p-nitrophenol (Figure 3).The p-nitrophenol substitution was made in an attempt to capture the putative cyclic intermediate by changing the rate of the spontaneous cyclization step (see Figure 2).The 31 P NMR spectrum of compound 1a is shown in Figure 4a.A single resonance is observed at ∼4.8 ppm for each diastereomer with a difference in chemical shift of ∼0.04 ppm.Similar results were obtained with compound 1b.The 31 P NMR spectrum of compound 1b has a pair of resonances at ∼4.1 ppm with a chemical shift difference of ∼0.03 ppm (Figure 5a).
Postulated Activation Mechanism.The proposed activation mechanism for the ProTide analogues synthesized for this investigation is presented in Figure 6. 1,6,7,9,10,12,14,15,24n the first step the isopropyl substituent of compound 1 is hydrolyzed by carboxypeptidase Y to form compound 2. The newly formed carboxylate spontaneously attacks the phosphorus center with expulsion of the phenol and formation of the putative cyclic intermediate 3. 6,12 In the final step, water attacks the mixed anhydride intermediate at either the phosphate or carboxylate center with the ultimate formation of compound 4. 6,10 For the enzyme-initiated transformation of 1 to 4, support for the postulated cyclic intermediate 3 has been reported, although the site of attack by water has not been determined for the formation of 4 from intermediate 3. 6,10,15,16 Hydrolysis of Compounds 1a and 1b by Carboxypeptidase Y.We utilized carboxypeptidase Y to initiate the hydrolysis of the isopropyl ester from compounds 1a/b and measured the release of either phenol or p-nitrophenol by monitoring the changes in the UV−vis spectrum as a function of time.The UV−vis spectrum of compound 1a is shown in Figure 7a, and that of compound 1b is in Figure 7b.After the addition of carboxypeptidase Y to compound 1a, there is a significant increase in the absorbance at 271 nm due to the ultimate formation of phenol (red spectrum in Figure 7a) at pH 8.0 (Δε 271 = 1,320 M −1 cm −1 ).For compound 1b, there is a significant increase in the absorbance at 400 nm after the addition of carboxypeptidase Y due to the formation of pnitrophenolate.However, the isosbestic point for p-nitrophenol is at 348 nm; therefore, reactions were monitored at this wavelength to avoid any variations due to pH (Δε 348 = 4,840 M −1 cm −1 ).Stereoselectivity of Carboxypeptidase Y for Hydrolysis of Compounds 1a and 1b.The stereoselective preference for the hydrolysis of the carboxylate ester in compounds 1a and 1b by carboxypeptidase Y was initially interrogated using 31 P NMR spectroscopy.After the addition of a small amount of carboxypeptidase Y (25 nM) to 4.0 mM of compound 1a, one of the two diastereomers is clearly hydrolyzed faster than the other (Figure 4b) with formation of intermediate 2a, depicted by the appearance of a new resonance at 5.54 ppm.Using a higher concentration of carboxypeptidase Y (200 nM), both diastereomers of intermediate 2a (new resonance at ∼5.40 ppm) are observed in Figure 4c, along with the complete hydrolysis of the faster diastereomer of compound 1a.The addition of a very high concentration of carboxypeptidase Y (3.9 μM) resulted in the rapid hydrolysis of both diastereomers of compound 1a and formation of nearly equal amounts of the two diastereomers of intermediate 2a, along with the ultimate formation of compound 4 (at 7.45 ppm) as shown in Figure 4d,e. No 3 P resonances were observed for the putative cyclic intermediate 3.
Carboxypeptidase Y also shows a stereoselective preference for the hydrolysis of compound 1b (Figure 5b) where the most upfield resonance at ∼4.10 ppm is diminished relative to the resonance for the other diastereomer.In this case, no new resonances are observed for either of the two diastereomeric intermediates (2b), indicating that the cyclization of intermediate 2b and expulsion of the p-nitrophenolate substituent is significantly faster than for the phenolate in intermediate 2a.However, the same ultimate product ( 4) is observed at 7.45 ppm in Figure 5b,c.The relative rates of ester hydrolysis of compounds 1a and 1b by carboxypeptidase Y were determined by monitoring the changes in the 31 P NMR spectra as a function of time after the addition of the enzyme to an equal mixture of compounds 1a and 1b.The time course is presented in Figure S1, and the relative rate of hydrolysis of the two compounds is ∼3.0.It is assumed here that the relative stereochemistry of the faster diastereomer is the same for compounds 1a and 1b, but the absolute stereochemistry of this isomer is unknown at this time.
Rate of Cyclization Determined by 31 P NMR Spectroscopy.The rate of cyclization of intermediate 2a was measured by following the formation of compound 4 and the disappearance of 2a by 31 P NMR spectroscopy after a large excess of carboxypeptidase Y was added to compound 1a (Figure 4d and Figure S2) and fitting the data to a single exponential using eq 1.The measured rate constant for the decay of compound 2a and the formation of compound 4 is 0.096 ± 0.005 min −1 , corresponding to a half-life of ∼7.2 min at 30 °C.A direct measurement for the cyclization of compound 2b could not be obtained due to the very rapid rate of p-nitrophenol release after hydrolysis of the carboxylate ester by carboxypeptidase Y (reaction is complete in <10 s) with an estimated rate constant of ≥10 min −1 .Therefore, 2b cyclizes ≥100-fold faster than 2a.
Mass Spectrometry Analysis.Electrospray ionization mass spectrometry (ESI-MS) was conducted in the positive ion mode for the initial identification of 1a, with an observed m/z for the [M+H] + cation at 316.1305 Da (Figure 8a).After the entire reaction sequence was complete, compound 4 was identified in the negative ion mode for the [M-H] − anion at 196.0371 Da (Figure 8b).When the reaction sequence was conducted in 90% 18 O-labeled water, the final product was identified by the [M-H] − anion at an m/z of 200.0475Da, indicating the incorporation of two oxygen-18 atoms from two water molecules in the conversion of 1a to 4 (Figure 8c).
The reaction products for the activation of compound 1b were also analyzed by ESI-MS.Compound 1b was first identified by the [M-H] − anion at an observed m/z of 359.1011Da (Figure 9a).After the addition of carboxypeptidase Y, the reaction products were identified in the negative ion mode at an m/z of 138.0182 (p-nitrophenolate) and compound 4 at 196.0371 Da (Figure 9b).The final reaction product, produced using 18 O-labeled water, is shown in Figure 9c with an m/z of 200.0455Da, indicating the incorporation of two oxygen-18 atoms from the reactions involving two separate water molecules.
Hydrolysis of Compounds 1a and 1b Monitored by UV−vis Spectroscopy.To further probe the reaction mechanism for activation of compound 1a by carboxypeptidase Y, the time course for the formation of phenol was monitored by UV−vis spectroscopy at 271 nm.Shown in Figure 10a are the time courses when different amounts of carboxypeptidase Y are added to compound 1a.At low enzyme concentrations there is a definite lag phase that reflects the differential rate of hydrolysis of the two diastereomers of 1a to 2a, and the relatively slow rate of cyclization of intermediate 2a to 4 via compound 3.At the highest concentration of carboxypeptidase Y, the time course can be depicted as following a single exponential with a rate constant of 0.058 ± 0.001 min −1 for the conversion of 2a to 3/4.This rate constant is in reasonable agreement to the value of 0.096 min −1 determined by 31 P NMR spectroscopy at a slightly higher temperature.
The time courses for the hydrolysis of compound 1b by carboxypeptidase Y are exhibited in Figure 10b.Since the rate constant for the cyclization of 2b to 3/4 is so much faster than for 2a to 3/4 these assays provide a continuous monitor for the differential rate of hydrolysis of the two diastereomers of 1b by carboxypeptidase Y.At the highest concentration of carboxypeptidase Y, the first phase is over within the dead-time of the assay, and at the lowest concentration of carboxypeptidase Y only a single phase is observed.However, at a concentration of 25 nM enzyme two approximately equal phases are observed and each phase could be fit to a single exponential with values of 1.39 ± 0.01 and 0.068 ± 0.001 min −1 , thus indicating that the faster diastereomer is hydrolyzed ∼20 times faster than the slower diastereomer (Figure S3).
Positional Hydrolysis of the Cyclic Intermediate.The hydrolysis of the putative cyclic intermediate 3 in the activation pathway shown in Figure 6 has two possible routes to product formation via either P−O or C−O bond cleavage.To determine the regiochemistry for product formation, the hydrolysis reactions were conducted using 18 O-labeled water and the possible product outcomes are illustrated in Figure 11 using compound 1a as an example.The initial hydrolysis of the isopropyl ester by carboxypeptidase Y in 18 O-labeled water will result in the incorporation of a single 18 O in the carboxylate product (5a).Cyclization of 5a with the expulsion of phenol will result in an equal mixture of 6 and 7 where the 18 O is  distributed in the bridging (6) and nonbridging positions (7).If the attack of water occurs at the phosphorus center of the cyclic intermediate, then a single product (8) will be formed with one 18 O-label attached to the phosphorus and the other attached to the carboxylate.Alternatively, if the attack of water occurs at the carboxylate center of the cyclic intermediate, then hydrolysis of 6 will produce 8, but hydrolysis of 7 will produce 9, where both 18 O-labels are found within the carboxylate moiety.ESI-MS demonstrated for the activation of both 1a and 1b in 18 O-labeled water that two 18 O atoms are incorporated in the final product (Figures 8c and 9c) but did not differentiate the specific site of 18 O-incorporation.
The product outcome for the activation of compound 1a was determined using 31 P NMR spectroscopy.It has been demonstrated previously that the substitution of an 18 Oisotope in phosphate esters will result in an upfield chemical shift difference of approximately 0.02−0.03ppm. 27,28Therefore, if a mixture of 8 and 9 were to be formed via the hydrolysis of the carboxylate ester, the 31 P NMR spectrum of the products would show two phosphorus resonances of equal intensity with a difference in chemical shift of ∼0.025 ppm.Alternatively, if 8 was exclusively formed via the attack of water on the phosphorus center then a single 31 P NMR resonance would be observed.The 31 P NMR spectrum of the final product when 1a is activated in 18 O-water is shown in Figure 12a.A single resonance is observed, demonstrating that water exclusively attacks the phosphorus center of the cyclic intermediate rather than the carboxylate center.To prove that the phosphate ester of the final product contains a single 18 O-label, the same reaction was conducted in unlabeled water (Figure 12b) and then the two products were combined in equal ratios.The 31 P NMR spectrum of the reaction mixture now shows two resonances separated by ∼0.03 ppm (Figure 12c).Again, this result demonstrates the attack at the phosphorus center in the putative cyclic intermediate formed during the activation of the ProTide analogue.An identical experiment was conducted using compound 1b and the overall conclusion is the same (the 31 P NMR spectra are presented in Figure S4).

■ CONCLUSIONS
ProTide pharmaceuticals have become an effective method for the treatment of viral infections.These compounds have a modified nucleotide core where the phosphate moiety is further esterified with phenol and amidated with a derivatized L-alanine.The design of these prodrugs requires in vivo modification to facilitate subsequent cellular phosphorylation.In the proposed mechanism of activation, a cellular protease/ esterase hydrolyzes a carboxylate ester from the L-alanyl moiety of the prodrug that subsequently cyclizes with the expulsion of the phenol.The putative cyclic intermediate then reacts with water to generate the phosphoramidate product.The nucleophilic attack of the cyclic intermediate could occur at either the phosphate or carboxylate center of the mixed anhydride intermediate.Here, we demonstrated that attack by water on the putative cyclic intermediate formed during the activation of ProTide analogues occurs exclusively at the phosphorus center.We also demonstrated that formation of the putative cyclic intermediate occurs more than 2 orders of magnitude faster with a p-nitrophenol derivative prodrug relative to one formed from phenol.

Figure 1 .
Figure 1.Chemical structures of three currently FDA approved ProTide pharmaceuticals.

Figure 2 .
Figure2.Proposed in vivo mechanism of activation for the ProTide Remdesivir.22,23In this mechanism, the cyclic intermediate can potentially be attacked by water at either the phosphate or carboxylate to generate the same product.

Figure 3 .
Figure 3. Analogues of ProTides synthesized for this investigation.The diastereomeric mixtures are racemic at the phosphorus center but chiral at the α-carbon of the L-alanine moiety.

Figure 4 .
Figure 4. 31 P NMR spectra of compound 1a at pH 8.0 and 30 °C.(a) Compound 1a prior to the addition of carboxypeptidase Y; (b) partial hydrolysis of compound 1a using 25 nM carboxypeptidase Y after 6 min; (c) partial hydrolysis of compound 1a using 200 nM carboxypeptidase Y after 6 min; (d) complete hydrolysis of 1a by 3.9 μM carboxypeptidase Y after 6 min; (e) final reaction product (4) formed with 200 nM carboxypeptidase Y after 30 min.

Figure 5 .
Figure 5. 31 P NMR spectra of compound 1b at pH 8.0 and 30 °C.(a) Compound 1b prior to the addition of carboxypeptidase Y; (b) partial hydrolysis of compound 1b by 25 nM carboxypeptidase Y after 10 min; (c) complete hydrolysis of compound 1b by 1.9 μM carboxypeptidase Y after 1 h.

Figure 6 .
Figure6.Working model for the activation of representative ProTides where X = H for 1a, and X = NO 2 for 1b.1,6

Figure 7 .
Figure 7. UV−vis spectra of compounds 1a (1.0 mM) and 1b (100 μM) before and after hydrolysis using 1.0 μM carboxypeptidase Y at pH 8.0 and 25 °C.(a) Compound 1a prior to the addition of carboxypeptidase (blue); compound 1a after the addition of carboxypeptidase Y (red).(b) compound 1b prior to the addition of carboxypeptidase Y (blue); compound 1b after the addition of carboxypeptidase (red).

Figure 8 .
Figure 8. Mass spectra for compound 1a and its hydrolysis products.(a) Positive ion ESI-MS of compound 1a ([M+H] + = 316.1305)prior to the addition of carboxypeptidase Y.(b) Negative ion ESI-MS for compound 4 ([M-H] − = 196.0371)after initiation of the hydrolysis of compound 1a with carboxypeptidase Y. (c) Negative ion ESI-MS for compound 4 ([M-H] − = 200.0475)after initiation of the hydrolysis of compound 1a with carboxypeptidase Y in 18 O-labeled water.

Figure 9 .
Figure 9. Mass spectra for compound 1b and its hydrolysis products.(a) Negative ion ESI-MS of compound 1b ([M-H] − = 359.1011)prior to the addition of carboxypeptidase Y.(b) Negative ion ESI-MS for compound 4 ([M-H] − = 196.0371)after initiation of the hydrolysis of compound 1b with carboxypeptidase Y. (c) Negative ion ESI-MS for compound 4 ([M-H] − = 200.0455)after initiation of the hydrolysis of compound 1b with carboxypeptidase Y in 18 O-labeled water.The hydrolysis product p-nitrophenol appears at an m/z of 138.0182 for the [M-H] anion in panels (b) and (c).

Figure 11 .
Figure 11.Possible reaction products for the activation of compound 1a in 18 O−H 2 O.

Figure 12 .
Figure 12. 31 P NMR spectra of the final reaction product when compound 1a is activated by carboxypeptidase Y. (a) Final product when the reaction was conducted in 18 O-water.(b) Final product when the reaction was conducted in 16 O-labeled water.(c) Mixture of the products shown in spectra a and b.
22,23In this mechanism, the cyclic intermediate can potentially be attacked by water at either the phosphate or carboxylate to generate the same product.